Regeneration of Salivary Gland Defects of Diabetic Wistar Rats Post Human Dental Pulp Stem Cells Intraglandular Transplantation on Acinar Cell Vacuolization and Interleukin-10 Serum Level

Objective: To investigate the regeneration of rat’s salivary gland diabetic defect after intraglandular transplantation of Human Dental Pulp Stem Cells (HDPSCs) on acinar cell vacuolization and Interleukin-10 (IL-10). Material and Methods: HDPSCs isolated from the dental pulp of first premolars #34. HDPSCs from the 3rd passage was characterized by immunocytochemistry of CD73, CD90, CD105 and CD45. Twenty-four male Wistar rats, 3month-old, 250-300 grams induced with Streptozotocin 30 mg/kg body weight to create diabetes mellitus (DM) divided into 4 groups (n=6); positive control group on Day-7; positive control group on Day-14; treatment group Day-7 (DM+5.105HDPSCs); treatment group on Day-14. On Day-7 and Day-14, rats were sacrificed. Histopathological examination performed to analyze acinar cells vacuolization while Enzyme-linked Immunoabsorbent Assay to measure IL-10 serum level. Data obtained were analyzed statistically using multiple comparisons Bonferroni test, Kruskal Wallis, Shapiro-Wilk and Levene’s test result. Results: The highest acinar cell vacuolization found in control group Day 14 (0.239 ± 0.132), meanwhile the lowest acinar cell vacuolization found in treatment group Day 7 (0.019 ± 0.035) with significant difference (p=0.003). The highest IL-10 serum level found in treatment group Day 14 (175.583 ± 120.075) with significant difference (p=0.001). Conclusion: Transplantation of HDPSC was able to regenerate submandibular salivary gland defects in diabetic rats by decreasing acinar cell vacuolization and slightly increase IL-10 serum level.


Introduction
Diabetes Mellitus (DM) is a progressive and chronic disease characterized by an increase in blood glucose levels. The prevalence of DM patients in the world was 8.8% and in Indonesia it was 6.2% [1]. Salivary gland defect is one of the complications of DM in the oral cavity. Based on a recent meta-analysis study, the global prevalence of xerostomia in DM patients is 42.4%. Salivary gland defects can reduce the patient's quality of life because it causes difficulty in swallowing, speaking, chewing, dysgeusia, caries and an increase in plaque accumulation [2]. Clinically, salivary gland defects are characterized by decreased salivary secretion volume and histologically characterized by acinar cell vacuolization, which is an early sign of cell degeneration [3,4].
Nowadays, salivary gland defects are treated by performing salivary stimulation either through oral receptors using ascorbic acid, stimulation of mastication by chewing gum, and by pharmacological stimulation using pilocarpine. However, all of these therapies cause various complications and are only temporary relief; therefore, long-term definitive therapy is still needed [5][6][7].
Bone Marrow Mesenchymal Stem Cells (BMMSC) which are considered to be a solution to problems in various tissue damage related to the tissue regeneration capacity by differentiating into various cell lines, having a paracrine effect and having immunomodulation capacity. However, the isolation of BMMSC is very invasive. Human Dental Pulp Stem Cells (HDPSC) are considered as a better source of MSC because their extraction is less invasive than BMMSC, possess the ability of self-renewal, higher plasticity, and proliferation compared to BMMSC, but has a similar pattern of gene expression, fenotip and protein in vitro with BMMSC [8][9][10][11].
DPSC allegedly has a good salivary gland regeneration capacity by being a component of epithelial cells (acinar) itself or by being a salivary gland mesenchymal component to induce and support the regeneration process of the salivary glands. Therefore, stem cell-based regenerative therapy is expected to produce replacement tissue that can mimic the natural structure and function of the salivary glands and provide long-term benefits for salivary gland defects [12][13][14][15].  μg / mL fungizone (A9528, Sigma Aldrich Corp., St. Louis, MO, USA). Cells were conditioned at a temperature of 37°C in 5% CO2 medium was replaced every 2-3 days. The cell culture was passaged three times to get the desired cell number [17][18][19].

Ethical
Immunocytochemistry Analysis HDPSC in the 3rd passages was then examined for cluster differentiation markers to ensure positive MSC markers, namely CD73, CD90, CD105 and negative CD45 markers. Cells were coated with coverslips, and after incubation at 37°C for 1-2 hours, fixation was done with 10% formaldehyde

Diabetic Rats Model
All animals (Wistar Rats) were housed in polycarbonate cages, subjected to a 12-hour lightdark cycle at the constant temperature of 23°C, and fed a standard pellet diet (expanded pellets; Stepfield, Witham, Essex, UK) with tap water ad libitum at a temperature of 22°C ± 2°C [21]. Mice are fastened for approximately 12 hours before induction to empty the stomach and accelerate the occurrence of DM conditions. Diabetic rats model was induced by STZ (Bioworld Merchandising Inc., Irving, TX, USA) with a dose of 30 mg / kg which was dissolved in citrate buffer (CV. Gamma Scientific Biolab, Malang, Indonesia) 30 mg/mL (pH 4.5) injected intraperitoneally in the area beside the midline between two nipples or in right / left mouse umbilication. Induction was done once; the mouse was held and the part to be injected is rubbed with 70% alcohol. The needle was injected perpendicular to the right / left umbilical to the peritoneal cavity; then the ingredients are injected slowly. Mice were given 10% sucrose solution or 10% dextrose (PT Otsuka, Jakarta, Indonesia) during the first night after induction to avoid sudden hypoglycemic post [22]. Mice were declared diabetic if on the 7th day after induction of blood sugar levels ≥ 200 mg / dL checked using Accu check (0197, EasyTouch GCU, Bioptik Technology Inc., Taiwan) [22,23].

HDPSC in Vivo Injection
HDPSC was injected in rats in the treatment group with a single dose of 5.10 5 cells / 250 gr BW in 0.2 mL intraglandular PBS solution in the submandibular gland. The control group mouse has injected 0.2 mL of PBS intraglandularly in the submandibular gland. The mice were sacrificed on the 7th and 14th day using rodent anesthesia (Ketamine 70 mg / kg BW and Xylazine 5 mL). The sub-mandibular gland and blood tissue were extracted for further analysis [19].

Paraffin Block Preparations
Preparations were obtained from salivary gland incisions, which were fixed with 10% neutral buffered formalin (NBF) (HT501128, Sigma Aldrich Corp., St. Louis, MO, USA) with solution volume of 10 times the size of the specimen. After fixation, dehydration was done through the extraction of water from the tissue with alcohol (30%, 50%, 70%, 80%, 96% and absolute) for 60 minutes each. Clearing was then carried out, which was to clear the tissue to be transparent by inserting the tissue into xylol solution 2 times each for 60 minutes. Impregnation was done consequently by infiltration with soft paraffin for 60 minutes at 48°C and went through block process in hard paraffin in the mold and let set for a day. Paraffin blocks were attached to the holder and were cut using a microtome rotary for 4-6 µm.

Staining and Interpretation of Hematoxylin and Eosin
Deparaffinization was done by submerging the sample in xylol for 5 minutes. The sample was re-hydrated using alcohol from high to lower concentrations (96%, 80%, 70%) for 2 minutes and at the end using water for 10 minutes. Staining was done with Mayer's Hematoxylin (MHS1, Sigma Aldrich Corp., St. Louis, MO, USA) for 15 minutes and washed with running water until samples appeared blue for 5 minutes or less. The samples were submerged in 1% acidic alcohol (1% HCL in 70% alcohol) for 5-10 seconds. Rinsing with water until appeared blue again for 10-15 minutes or submerse in alkaline solution (ammonia water) followed by washing for 5 minutes. The sample was stained with 1% eosin Y for 10 minutes. Wash it with water for 1-5 minutes. The sample was dehydrated with alcohol 70%, 80% and 96% for 2 minutes of each. Clearing was performed by

Results
After isolation and culture, DPSC was the passage for 3 times to obtain desired cell numbers     There were significant differences between K+14 and K+7, P+7 and K+14, P+14 and K+14 group (p<0.05) ( Table 2). The IL-10 serum level data obtained were not homogenous and not normally distributed (p<0.05) with a significant difference (p=0.001, p<0.05).

Table 2. Multiple Comparisons result of acinar cells vacuolization between groups.
The highest level of IL-10 serum was found in the treatment group on the 14th day (175.583 ± 120.075), meanwhile the lowest level of IL-10 serum found in the control group on the 7th day (24.071 ± 9.316) ( Table 3). There were significant differences between groups (p=0.001, p<0.05). There were significant differences between K+7 and K+14, K+7 and P+14, P+7 and P+14 groups (p<0.05) ( Table 4).

Discussion
In this present study found that acinar cell vacuolization between the control and treatment groups Day 7 were not significantly different. This is indicated that the tissue regeneration process by DPSC on the 7th day had not occurred well. This is supported by previous research that that on the 7th day of the HDPSC injection post there were no significant results on the microvascular structure, which is one of the important factors in the regeneration process [24]. Acinar cells vacuolization tends to increase in both groups Day 7 and Day 14 due to blood glucose did not controlled; therefore, the progressive diabetic complication worsens each day.

Acinar cells vacuolization between the control and treatment groups
However, the acinar cells vacuolization increased significantly in the control group than the treatment group. This indicated that HDPSC only able to decrease and inhibit the progression of acinar cells vacuolization.
The literature shows that IL-10 inhibits macrophage activation [26]. Meanwhile, MSC will increase IL-10 expression through macrophages, dendritic cells, and peripheral blood mononuclear cells [27]. It means that the administration of MSC will increase IL-10 levels comparable to levels of macrophages in serum.
The elevated IL-10 levels in control group will reduce the levels of existing macrophages, so that the administration of MSC increases in IL-10 levels did not appear to be too significant. This can explain why in this experiment, there was no significant increase in the treatment group when compared with the control group both on day 7 and 14. This result did not in accordance with the previous study, which said that DPSC was able to significantly increase IL-10 production when DPSC was conditioned with Peripheral Blood Mononuclear Cells (PBMSC) [28].
It has been shown that DPSC transplanted in the sciatic nerve was able to decrease IL-10 levels in the sciatic nerve sample [29]. When compared between this study and others [28,29], the results of the study showed that there were no differences between the control group and treatment groups could occur due to differences in the injection location of DPSC and the samples used for IL-10 levels, namely DPSC injected intraglandularly in the submandibular gland while the IL-10 examination sample originated from peripheral blood serum.
However, the effect may be seen systemically if the dose from DPSC is increased as in an experiment carried out previously [30], which demonstrated that expression of IL-10 serum levels would recover in repeated transplants from DPSC at a dose of 1x10 6 cells intra-muscular. In this experiment, only single-dose DPSC was used (5x10 5 cells intraglandularly). Hence, the absence of significant differences in this study could be due to the dose being too low and not being given repeatedly dose of DPSC, which caused intraglandular administration could not provide a systemic effect on serum levels of IL-10.

Conclusion
Transplantation of HDPSC was able to regenerate submandibular salivary gland defects in diabetic rats by decreasing acinar cell vacuolization and slightly increase IL-10 serum level.

Conflict of Interest:
The authors declare no conflicts of interest.